Downregulation of E-cadherin in pluripotent stem cells triggers partial EMT

Abstract

Epithelial to mesenchymal transition (EMT) is a critical cellular process that has been well characterized during embryonic development and cancer metastasis and it also is implicated in several physiological and pathological events including embryonic stem cell differentiation. During early stages of differentiation, human embryonic stem cells pass through EMT where deeper morphological, molecular and biochemical changes occur. Though initially considered as a decision between two states, EMT process is now regarded as a fluid transition where cells exist on a spectrum of intermediate states. In this work, using a CRISPR interference system in human embryonic stem cells, we describe a molecular characterization of the effects of downregulation of E-cadherin, one of the main initiation events of EMT, as a unique start signal. Our results suggest that the decrease and delocalization of E-cadherin causes an incomplete EMT where cells retain their undifferentiated state while expressing several characteristics of a mesenchymal-like phenotype. Namely, we found that E-cadherin downregulation induces SNAI1 and SNAI2 upregulation, promotes MALAT1 and LINC-ROR downregulation, modulates the expression of tight junction occludin 1 and gap junction connexin 43, increases human embryonic stem cells migratory capacity and delocalize β-catenin. Altogether, we believe our results provide a useful tool to model the molecular events of an unstable intermediate state and further identify multiple layers of molecular changes that occur during partial EMT.

Figure 1

Characterization of HES3-KRAB^CDH1 clonal cell line. (a) Schematic representation showing the construct used for generation of HES3-KRAB^CDH1 cell line. This knockdown system contains a deactivated Cas9 (dCas9) fused to a transcriptional repressor domain Krüppel Associated Box (KRAB) under the control of TRE promoter inducible by Dox. A sgRNA against CDH1 promoter was placed under the control of the U6 promoter which is constitutively activated. In cells not treated with Dox, the system is not active. Upon Dox induction, dCas9-KRAB is expressed and targeted to CDH1 promoter by the sgRNA to produce transcriptional repression of E-cadherin. (b) Relative mRNA levels of E-cadherin (CDH1) in cells after incubation with Dox at different times assessed by qPCR. AU is the abbreviation for arbitrary units. Results are represented as mean ± SD (n = 5). Different letters indicate significant differences of groups compared to control condition (p < 0.001) for ANOVA with post hoc Tukey. (c) Left panel: immunofluorescence images of E-cadherin protein (green) in cells incubated with Dox during 48, 72 and 96 h. Nuclei were stained with DAPI (blue). Magnification of the indicated areas are shown in black and white at the bottom of the panel. Representative images of at least three experiments are shown. Scale bar 50 µm. Right panel quantification of the mean fluorescence intensity of three images from three independent experiments. Results are represented as mean ± SD (n = 3). Different letters indicate significant differences of groups compared to control condition (p < 0.0001) for ANOVA with post hoc Tukey.

Figure 2

Downregulation of E-cadherin induced morphological alterations and transcriptional regulation of some adhesion complex genes. a) Morphological changes of HES3-KRAB^CDH1 cells incubated with Dox at different times. Cells were stained with phalloidin (grey) to visualize F-actin and DAPI was used to stain the nucleus (blue). Magnifications of areas indicated by dotted lines are shown in the last column in black and white colors. Scale bar 50 µm. b) Cellular size and shape analysis of HES3-KRAB^CDH1 cells. Cell membranes were defined by wheat germ agglutinin (WGA) labeling after incubation without Dox (Control) or with Dox during 96 h (96 h). Upper panel: representative images of cell membrane stained with WGA (green). Nucleus were stained with DAPI (blue). Scale bar 50 µm. Lower panel: graphical representation of quantification of cell area, perimeter and circularity. AU is the abbreviation for arbitrary units. (c) Relative mRNA expression of desmoplakin (DSP), desmoglein (DSG2), desmocollin (DSC2) (d) tight junction protein 1 (TJP1), claudin 3 (CLDN3), occludin 1 (OCLN) and (e) connexin 43 (GJA1), when E-cadherin is downregulated. AU is the abbreviation for arbitrary units. Results are represented as mean ± SD (n = 5). ANOVA with post hoc Tukey. Different letters indicate significant differences of groups compared to control condition (p < 0.05). (f) Left: normalized mRNA expression of E-cadherin (CDH1), occludin-1 (OCLN) and connexin 43 (GJA1) genes. Right: heat map showing Pearson's correlation coefficient between these genes.

Figure 3

E-cadherin decrease does not modify pluripotency state. (a) qPCR analysis of pluripotency genes OCT4, NANOG and LIN28 after Dox treatment of HES3-KRAB^CDH1 cells at different times. AU is the abbreviation for arbitrary units. Results are represented as mean ± SD (n = 5). ANOVA with post hoc Tukey. Different letters indicate significant differences of groups compared to control condition (p < 0.05). (b) Immunofluorescence and quantification of mean fluorescence intensity of OCT-4 (upper panel), SOX2 (middle panel) and NANOG (lower panel) (red) in HES3-KRAB^CDH1 cells. Nucleus were stained with DAPI (blue). Scale bar 100 µm. (c) Expression of NANOG and OCT-4 (pluripotency genes), GATA4 (endoderm gene), NKX2.5 (mesoderm gene) and TUBB3 (ectoderm gene) in HES3- KRAB^CDH1 cells and embryoid bodies (EB) with or without Dox treatment for 96 h. AU is the abbreviation for arbitrary units. Results are represented as mean ± SD.

Figure 4

E-cadherin decrease activates the initiation of EMT. qPCR analysis of (a) genes involved in EMT such as SNAI1, SNAI2, ZEB1 and ZEB2 and (b) genes of early mesoderm precursor TBX6 and MIXL1 when E-cadherin is downregulated. (c) Relative expression level of LINC-ROR and MALAT1, two lnc-RNAs that act as regulators of EMT were measured by qPCR. AU is the abbreviation for arbitrary units. Results are represented as mean ± SD (n = 5). ANOVA with post hoc Tukey. Different letters indicate significant differences of groups compared to control condition (p < 0.05). (d) Immunofluorescence and quantification of mean fluorescence intensity of β-catenin (grey) in cells treated with Dox. Nuclei were stained with DAPI (blue). Magnification of dotted areas are shown in black and white colors. Scale bar 50 µm. Representative images of at least three experiments are shown. Results are represented as mean ± SD (n = 3). ANOVA with post hoc Tukey. Different letters indicate significant differences between groups (p < 0.05). (e) Relative mRNA levels of c-myc (MYCBP) and cyclin D1 (CCND1) were determined by qPCR. Data are presented as mean ± SD (n = 4). ANOVA with post hoc Tukey. Different letters indicate significant differences of groups compared to control condition (p < 0.05).

Figure 5

Silencing of E-cadherin induced a partial EMT state. (a) Wound healing assay was made after cells were treated with or without Dox (+Dox and −Dox, respectively) (t = 0 h). Cells were treated with Dox during 72 h and then scratch was made (t = 0 h for this experiment). Images were taken at t = 0 h and after 22 h of recovery (t = 22 h) (left). Graphic represents the relative wound closure area of at least four independent experiments (right). AU is the abbreviation for arbitrary units. T test. Different letters indicate significant differences. (b) Rescue experiment to regain the knockdown effect. Cells were incubated with Dox for 96 h. At this time point, Dox was removed and cells were maintained for another 96 h (4 days) only with culture medium. CDH1 and SNAI1 gene expression was evaluated by qPCR. Results are represented as mean ± SD (n = 5). AU is the abbreviation for arbitrary units. ANOVA with post hoc Tukey. Different letters indicate significant differences of groups compared to control condition (p < 0.01).